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Decision Document DD96-08:
Determination of Environmental Safety of
Calgene Inc.'s High Laurate Canola
(Brassica napus L.)
REVISED

This Decision Document has been prepared to explain the regulatory decision reached under the guidelines Dir94-08 Assessment Criteria for Determining Environmental Safety of Plants with Novel Traits and its companion document Dir94-09 The Biology of Brassica napus L. (Canola / rapeseed), and the guidelines Dir95-03 Guidelines for the Assessment of Livestock Feed from Plants with Novel Traits.

The Canadian Food Inspection Agency (CFIA), specifically the Plant Biosafety Office of the Plant Health and Production Division and the Feed Section of the Animal Health and Production Division, has evaluated information submitted by Calgene Inc. regarding two transformed canola lines referred to as high laurate canola in the present document. These plants were transformed with a gene resulting in production of higher contents of laurate in the seed oil, and a gene conferring resistance to kanamycin as a selectable marker. CFIA has determined that these plants with novel traits should not pose concern to environmental safety.

Unconfined release into the environment, including feed use as meal, of the High Laurate canola lines 23-198 and 23-18-17, and other B. napus lines derived from them, but without the introduction of any other novel trait, is therefore considered safe.

* In the revised document, the term high laurate rapeseed was changed to high laurate canola.

Table of Contents

1. Brief Identification of the Plants with Novel Traits (PNT's)
2. Background Information
3. Description of the Novel Traits
   1. Production of High Levels of Laurate in Seed Oil
   2. Kanamycin Resistance
   3. Development Method
   4. Stable Integration into the Plants' Genomes
4. Assessment Criteria for Environmental Safety
   1. Potential of the PNT's to Become Weeds of Agriculture or Be Invasive of Natural Habitats
   2. Potential for Gene Flow to Wild Relatives Whose Hybrid Offspring May Become More Weedy or More Invasive
   3. Altered Plant Pest Potential
   4. Potential Impact on Non-Target Organisms
   5. Potential Impact on Biodiversity
5. Nutritional Assessment Criteria for Use as Livestock Feed
   1. Anti-Nutritional Factors
   2. Nutritional Composition of the PNT's
6. Regulatory Decision

I. Brief Identification of the Plants with Novel Traits (PNT's)

II. Background Information

Calgene Inc. has developed two canola lines producing high lauric acid levels in the seed. These lines, referred to as high laurate canola (lines 23-198 and 23-18-17) in the present document, will provide an alternative for exotic sources of laurate oil (e.g. palm kernel oil) to be used in food products, animal feed, or industrial applications such as laundry detergent.

The development of the high laurate canola lines was based on recombinant DNA technology, by the introduction of a plant gene and a bacterial gene into the B. napus cultivar 212/86. The plant gene codes for an enzyme in the fatty acid biosynthetic pathway of the developing seeds, whose activity results in the release of the saturated fatty acid laurate. The other gene, conferring resistance to kanamycin, is of no agronomic interest but was used to select modified plants from those that remained unmodified at the development stage.

These lines have been tested in Canada under confined conditions since 1993 in Alberta (1993-95), Saskatchewan (1993-95), Manitoba (1993-95), and Ontario (1994). They have received complete approval for commercialization in the United States.

Calgene Inc. has provided data on the identity of high laurate canola, a detailed description of the transformation vector and the modification method, data and information on the number of gene insertion sites, integrity of gene integration, copy numbers and expression in the plant, the role of the inserted genes and regulatory sequences in donor organisms, segregation analyses and full nucleotide sequences.

The novel proteins were identified and characterized, including a description of breakdown products and potential impact on metabolic pathways, and a discussion of their potential toxicity to interacting organisms. A large number of scientific publications was also supplied.

Agronomic characteristics such as overwintering capacity, seed germination, seedling vigour, stand establishment, plant vigour, fertility, flowering period, time to seed maturity, seed yield, shattering, lodging and harvest stand, were compared to those of unmodified B. napus counterparts. Stress adaptation was evaluated, including susceptibilities to various pathogens and insects.

Composition analyses of the seed were performed, including proteins, fatty acid profiles of the oil, and endogenous toxins.

The Plant Biosafety Office, has reviewed the above information, in light of the assessment criteria for determining environmental safety of plants with novel traits, as described in the regulatory directive Dir94-08:

• potential of the PNT's to become weeds of agriculture or to be invasive of natural habitats,
• potential for gene-flow to wild relatives whose hybrid offspring may become more weedy or more invasive,
• potential for the PNT's to become plant pests,
• potential impact of the PNT's or their gene products on non-target species, including humans, and
• potential impact on biodiversity.

The Feed Section has also reviewed the above information in light of the assessment criteria for determining the safety and efficacy of livestock feed, as described in Dir95-03:

• potential impact to livestock, and
• potential impact on livestock nutrition.

III. Description of the Novel Traits

1. Production of High Levels of Laurate in Seed Oil

• A gene from the California bay (Umbellularia californica) was introduced into the B. napus cultivar 212/86. This gene codes for a thioesterase (Bay TE), an enzyme active in the fatty acid biosynthetic pathway of the developing seed.
• The gene is linked to a seed specific promoter, active only in developing seed embryos. As a consequence, expression occurs only in maturing seeds. Dry seeds and any other plant parts have immunologically undetectable levels of the enzyme.
• Gene expression results in the production of a 382 amino acid long preprotein that is transported to the stroma of plastids where a 60 amino acid transit peptide is removed to produce the mature Bay TE enzyme. This enzyme accounts for 0.015% of the total extractable protein at mid-maturation of the developing seed.
• Studies showed that the introduced Bay TE enzyme is not heat stable and is fully degraded into inactive peptides and amino acids following protease digestion. Two potential glycosylation sites were identified but glycosylation of plastid proteins is not known to occur. Thioesterases are ubiquitous in plants and animals and Bay TE is not expected to be toxic or allergenic to non-target organisms. When subjected to comparative analyses with genetic sequence databases, no significant homology was found with any known sequenced toxins or allergens, whereas significant homology was found with thioesterases from edible plant species.
• The full nucleotide sequence of the gene was provided.
• Activity of the Bay TE results in the accumulation of the saturated fatty acid laurate (12:0), and to a lesser extent, of the saturated fatty acid myristate (14:0), in triacylglycerol molecules of seed oil, at the expense of oleic acid and linoleic acid.

2. Kanamycin Resistance

• Kanamycin is an aminoglycosidic antibiotic that binds to bacterial ribosomes thus disrupting normal protein synthesis and killing the bacterial cell.
• The kanamycin-resistance gene, isolated from the bacterium Escherichia coli, codes for an enzyme that prevents kanamycin from binding to ribosomes, through phosphorylation, thereby rendering the cells resistant. It is very specific and is not known to affect any plant metabolic pathway.
• The gene is linked to a strong constitutive promoter. Dry seeds have immunologically undetectable levels of the enzyme.
• The enzyme is ubiquitous in the environment. It degrades rapidly in simulated mammalian gastric and intestinal fluids.
• The nucleotide sequence showed no significant homology with the toxins or allergens entered in the GENEBANK DNA database. Toxic properties are not expected since: (1) the enzyme catalyses only a very specific reaction using aminoglycosidic antibiotics as a substrate, (2) despite wide usage, there have been no reports of toxicity of the protein in the literature and (3) enzyme levels are less than 0.0008% of total protein in seeds and leaves, and undetected in meal.
• The full nucleotide sequence of the gene was provided.
• The presence of the kanamycin resistance gene in livestock feed should not compromise the efficacy of added antibiotics. Neomycin activity when mixed with meal from High Laurate canola remained stable for a period of 56 days, and was not different as compared to the control line.

3. Development Method

• The B. napus cultivar 212/86 was transformed using a disarmed non-pathogenic Agrobacterium tumefaciens binary vector; the transferred construct contain the T-DNA region of an Agrobacterium plasmid from which virulence and plant disease-causing genes were removed, and replaced with genes coding for laurate production and kanamycin resistance. The T-DNA portion of the plasmid is known to insert randomly into the plant's genome and the insertion is usually stable, as was shown to be the case in high laurate canola.

4. Stable Integration into the Plants' Genomes

• The provided data showed that there was no incorporation of any coding region from outside the T-DNA borders. The original transformation event 23 was estimated to have 15 copies of the genes, at five independent genetic loci, as shown by Southern and segregation analyses.
• Lines 23-198 and 23-18-17 are several generations removed from the original transformant. Mendelian inheritance, and Southern and PCR analyses of third-generation material show the stability of the introduced genes as the bands did not change position. Some of the copies are segregating out, as expected, so that these lines may not be homogeneous for the number of copies present.

IV. Assessment Criteria for Environmental Safety

1. Potential of the PNT's to Become Weeds of Agriculture or Be Invasive of Natural Habitats

CFIA has evaluated the data and information submitted by Calgene Inc. on the reproductive and survival biology of the canola lines 23-18-17 and 23-198. From these, CFIA has determined that seed germination, seed yield, seedling growth, flowering and maturity dates were within the normal range of expression currently displayed by unmodified varieties. The laurate canola lines are generally taller, later maturing and lower yielding than Canadian adapted varieties, reflecting characteristics of the parent cultivar 212/86. Seed burial studies showed that the laurate canola lines are not persistent in soils of Western Canada and laboratory experiments showed that dormancy potential was not altered. Disease incidence (i.e. stem rot, blackleg, black spot, black rot, powdery mildew, gray stem, damping off, turnip mosaic, cauliflower mosaic, crown gall) and insect susceptibilities (flea beetles and aphids) were monitored during field trials and in greenhouses and were shown not to be different from those of unmodified counterparts.

High laurate canola lines have no specific added genes for cold tolerance or winter hibernation; no overwintered plants were observed by Calgene Inc. in post-harvest years of field trials in Canada.

The biology of Brassica napus, described in Dir94-09, shows that unmodified plants of this species are not invasive of unmanaged habitats in Canada. According to the information and data provided by Calgene Inc., high laurate canola lines were determined not to be different from their counterparts in this respect.

No competitive advantage was conferred to the lines 23-18-17 and 23-198. The modification of the seeds fatty acid composition will not render these lines weedy or invasive of natural habitats, since their reproductive characteristics were not modified. High laurate canola volunteers can be managed using common cultivation practices.

The above considerations, together with the fact that the novel traits have no intended nor unintended effects on weediness or invasiveness, led CFIA to conclude that the lines 23-18-17 and 23-198 have no altered weed or invasiveness potential compared to currently commercialized B. napus varieties.

2. Potential for Gene Flow to Wild Relatives Whose Hybrid Offspring May Become More Weedy or More Invasive

Brassica napus plants are known to outcross up to 30% with other plants of the same species, and potentially with plants of the species B. rapa, B. juncea, B. carinata, B. nigra, Diplotaxis muralis, Raphanus raphanistrum, and Erucastrum gallicum, found in disturbed habitats (see Dir94-09). Studies show that potential introgression of the genes is most likely to occur with B. rapa, the other major canola species, which occasionally is a weed of cultivated land, especially in the eastern prairie provinces of Canada.

If canola individuals expressing high levels of laurate did arise through interspecific or intergeneric hybridization, the traits would not confer any competitive advantage to these plants and these individuals would be controlled using common cultivation practices.

The above considerations led CFIA to conclude that gene flow from lines 23-18-17 and 23-198 to relatives is indeed possible, but would not result in increased weediness or invasiveness of these relatives.

3. Altered Plant Pest Potential

The intended effects of both novel traits are unrelated to plant pest potential, and B. napus is not a plant pest in Canada. In addition, agronomic characteristics, stress adaptation and quantitative and qualitative composition of the plants (excluding seeds) were shown to be within the range of values displayed by currently commercialized B. napus varieties. No unusual nutrient depletion from the soil is expected, as yield is lower than that of the unmodified parental line.

CFIA has therefore determined that plant pest potential of these lines had not been inadvertently altered.

4. Potential Impact on Non-Target Organisms

The detailed characterization of the novel genes, resulting enzymes, and modified fatty acid profiles of the seed, as briefly summarized in Part III of the present document, has led to the conclusion that these do not result in altered toxic or allergenic properties and that no adverse effects on non-target organisms are to be expected. The enzymes are ubiquitous in plants and animals, and are rapidly inactivated in mammalian stomach and intestinal fluids by enzymatic degradation and pH-mediated proteolysis. The Bay TE is only produced in the developing seed and the gross composition of the plant green matter is unchanged. High laurate is produced at high levels in common food sources, such as coconuts, palm oil, and mammalian milk. Laurate is used in food products.

The concentration of the native toxins erucic acid and glucosinolates are within acceptable levels for canola quality, and the quality and quantity of seed protein is as expected. The intended trait was a modification of the seed oil fatty acid profile. The overall lipid contents of the seed remained unchanged, with increased levels of lauric acid (up to 40%) and myristic acid, and lower levels of oleic acid and linoleic acid, and less palmitic acid, less stearic, linoleic, arachidic, gadoleic, and slightly more behenic acid. High laurate canola does not meet the specification for canola oil in the Food Chemicals Codex (1992) and should not be mixed with regular canola seed to be used for canola oil production. The high laurate B. napus lines will be grown under contract to preserve their identity and prevent mixing with conventional canola.

Based on the above, CFIA has determined that, when compared with currently commercialized counterparts, the unconfined release of the high laurate canola lines will not result in altered interactions with other organisms, including humans.

5. Potential Impact on Biodiversity

Lines 23-18-17 and 23-198 have no novel phenotypic characteristics which would extend their use beyond the current geographic range of B. napus production in Canada. Data and information submitted by Calgene Inc. have demonstrated to CFIA that these lines are not invasive of natural habitats, and that they are not more competitive than their counterparts, both in natural and managed ecosystems. Since outcross species are only found in disturbed habitats, transfer of the novel genes would have no impact on unmanaged environments.

CFIA has, therefore, concluded that the potential impact on biodiversity of high laurate lines is equivalent to that of currently commercialized B. napus varieties.

V. Nutritional Assessment Criteria for Use as Livestock Feed

1. Anti-Nutritional Factors

The glucosinolate content of the meal was not significantly different from any of the 10 control lines. Erucic acid levels in oil were less than 0.1% in test varieties.    High laurate canola contained levels of anti-nutritional factors below the prescribed standards for both the meal and/or oil fractions, i.e., <30 micromoles glucosinolates per gram of dry meal and <2% erucic acid in the oil.

2. Nutritional Composition of PNT

No statistical differences in crude protein, crude fibre, gross energy content, and amino acids were noted between the processed meal of high laurate and control B. napus cultivars. Ash content was not significantly different than the parental line (212/86). As expected, the fatty acid profile of high laurate canola was different from all control varieties. Laurate and myristate make up 40% and 4% of the fatty acid content of high laurate canola oil. Based on a maximum of 4% oil in the meal, this translates into 1.6% laurate and 0.16% myristate in meal. As the use rate of meal in livestock rations ranges from 5-25%, the maximum laurate and myristate in meal would be 0.4% and 0.94% respectively. This feeding rate would have insignificant impact on nutrition or carcass quality. The safety evaluation did not include an assessment of the use of the high laurate specialty oil as livestock feed since there is no intention to market the oil for this purpose.

Meal derived from lines 23-18-17 and 23-198 is judged to be substantially equivalent to meal from traditional B. napus varieties in terms of nutritional composition and safety.

VI. Regulatory Decision

Based on the review of Calgene Inc.'s data and information submitted to the Plant Biosafety Office, CFIA concludes that the novel genes of lines 23-18-17 and 23-198, and their corresponding novel traits do not in themselves confer any intended ecological advantage to the plant or to its relatives, should gene flow occur. The characteristics of these lines with regards to the assessment criteria for environmental safety are not altered. Thorough comparisons of the high laurate canola lines and unmodified B. napus counterparts showed no unexpected effects.

Based on the review of data submitted to the Feed Section, CFIA concludes that the novel genes of lines 23-18-17 and 23-198, and their corresponding traits do not in themselves raise any concerns regarding livestock safety or the nutritional composition of these lines. Canola oil and meal are currently listed in Schedule IV of the Feeds Regulations and are, therefore, approved for use in livestock feeds in Canada. Lines 23-18-17 and 23-198 have been assessed and the meal has been found to be substantially equivalent to meal of traditional canola varieties. It, therefore, meets the present ingredient definition of canola meal and is approved for use as canola meal in Canada. Oil derived from these lines should not be utilized as livestock feed at this time.

If at any time, Calgene Inc. becomes aware of any new information regarding risk to the environment, or risk to animal or human health, that could result from release of these materials in Canada, or elsewhere, Calgene Inc. must immediately provide such information to CFIA. On the basis of such new information, CFIA may re-evaluate the potential impact of the release and re-evaluate its decision.

Unconfined release into the environment, including feed use as meal, of the high laurate canola lines 23-18-17 and 23-198, and other B. napus lines derived from it, provided no other novel traits are introduced, is therefore considered safe.

This bulletin is published by the Plant Health and Production Division. For further information, please contact the Plant Biosafety Office or the Feed Section at: